The ability to adapt to hypoxic stress is crucial to the survival of virtually all life forms. These adaptations are mediated largely by changes in gene expression. One family of hypoxia-inducible genes, including erythropoietin (Epo), vascular endothelial growth factor (VEGF), and the glucose transporters GLUT1 and GLUT3, has also been shown to be upregulated by the transition metals cobalt, nickel, and manganese. Little is known about the oxygen (02) sensing pathway(s) which mediate this regulation. The long term objective of this proposal is to clone and characterize the genes which play a significant role in the 02 sensing pathway(s) of mammalian cells. This has proven difficult to accomplish in diploid eukryotic cells. Given the paramount importance of the adaptive responses to hypoxia, it seemed reasonable to hypothesize that these responses are highly conserved. We have shown that when the yeast, Saccharomyces cerevisiae, is transformed with a reporter construct consisting of the promoter of the anaerobically-induced yeast gene, ANB1, attached to the lacZ gene and grown under hypoxic conditions or in the presence of the transition metals mentioned above, an inducible phenotype is observed which is similar to the hypoxia and transition metal induction of Epo, VBGF, GLUT1, and GLUT3. Moreover, disruption of a candidate 02 sensor in yeast recapitulates this hypoxic phenotype even under normoxic conditions. These findings provide a new strategy to identify the genes involved in the 02 sensing pathway. The specific aims of this proposal are: 1) To identify the S. cerevisiae gene(s) responsible for the phenotype observed with the ANB1-lacZ reporter construct and confirm its hypoxia and cobalt inducibility, analogous to mammalian genes. 2) To use the gene(s) identified in Specific Aim #1 in assays designed to identify the genes involved in the 02 sensing pathway in & cerevisiae using standard yeast genetic techniques. 3) To further test the hypothesis that the recently cloned FHB gene encodes an 02 sensor in S. cerevisiae. 4) To identify the homologous genes involved in 02 sensing and signal transduction in mammalian cells using complementation strategies, low stringency hybridization of mammalian DNA libraries, and computer searches. Acquiring a better understanding of the molecular mechanisms governing the response to hypoxic stress is the crucial first step in the design and development of novel strategies to treat human conditions resulting from, or exacerbated by, a lack of 02. Likely applications would be aimed at improving the ability to treat anemias as well as vascular and neurological diseases.